Fig. 7.

Triple staining of unilateral ureteral obstruction (UUO) kidneys. F4/80 expression was enhanced in UUO kidneys of endothelial sirtuin 1-deficient (Sirt1^endo−/−) mice. A: representative images of F4/80- and syndecan-4 (Synd4)-stained contralateral (CLK) and UUO kidney sections from wild-type (WT) and Sirt1^endo−/− mice (n = 4). Scale bar = 10 µm. No permeabilization was used. B: quantification of F4/80 intensity per cell. A F4/80-stained image was merged with the respective Hoechst-stained image. Cells with F4/80-positive staining were counted manually. Finally, the measured total integrated density of F4/80 staining was divided by the respective total amount of F4/80-positive cells. Horizontal lines indicate statistical significance (P < 0.05). C: costaining of Synd4 in F4/80-positive cells. After the total amount of F4/80-positive cells was counted, as previously described, the respective Synd4-stained image was merged with the already merged F4/80/Hoechst image. Cells with F4/80/Synd4-positive staining were counted manually plus cells with Synd4 staining only. With the total amount of F4/80-positive and Synd4-positive cells, the percentage of the costained cells was calculated.